Problems making chemically competent cells

[lilruthc]

Hi all, I realise this is probably asked quite a bit so I apologise in advance!

I am looking to trouble shoot my competent cell making process. I make competent cells for a number of labs, but struggle to get the transformation efficiency of chemically competent cells above 10^7 (I get say 2 x 20^7 at best). I am very careful to ensure everything is cold. Any help here would be appreciated.

I've pasted the exact method I use below this:

• Pre-chill a flask on ice for the chilling step
• Pre-chill the centrifuge and its rotor
• Work in the cold room!
• Pre-chill eppendorfs in the -70 freezer, and tips in the cold room, (and maybe an eppendorf rack in the -80)

1. Make a 5ml pre-culture overnight from a single colony, in SOC medium
2. The next day, inoculate 100ml pre-warmed SOC medium with 5ml of the overnight pre-culture. The volume of the flask here is important for aeration. I use a 2 litre baffled flask for improved aeration (volume of medium 1/10 or 1/30 volume of the flask).
3. Incubate the culture at 37°C with vigorous shaking until the OD600 reaches 0.4-0.6 (4-7x107 cells/ml).
4. When at the correct OD, chill the culture on ice for 10-15 minutes. I use a pre-chilled flask for this in a wet ice bath.
5. Spin the culture at 3000 RPM for 15 minutes at 4°C in a pre-chilled rotor.
6. Resuspend the pellet in RF1 solution. The volume should be 1/3 of the culture volume
7. Incubate the cell suspension on ice for 15 minutes.
8. Pellet the cells as in step 5.
9. Resuspend the pellet in 8ml (1/12.5 of the original culture volume) of RF2.
10. Incubate the cell suspension on ice for 15 minutes.
11. Aliquot the cells into pre-chilled eppendorfs.
12. (Immediately flash freeze the aliquots in liquid nitrogen, then store at -70°C.)

Solutions:
RF1
Compound (end concentration) Amount for 100ml
RbCl (100mM) 1.2g
MnCl2•4H2O (50mM) 0.998g
Potassium acetate (30mM) 3ml (1M stock pH 7.5, 9.814g in 100ml solution, pH with acetic acid)
CaCl2•2H2O (10mM) 0.15g
Glycerol (15% w/v) 15g
Final pH: 5.8 with dilute acetic acid. Don’t overshoot – if you do, start again

RF2
Compound (end concentration) Amount for 100ml
RbCl (10mM) 0.12g
MOPS (10mM) 2ml (stock solution 0.5M, pH6.8 with NaOH, 10.46g in 100ml solution, adjust pH with NaOH)
CaCl2•2H2O (75mM) 1.1g
Glycerol (15% w/v) 15g
Final pH 6.8 with NaOH

[lilruthc]

Oh the one thing I am thinking of trying is Millipore water (I currently use autoclaved RO water for my RF1 and RF2) - could this be a big problem?